A novel tricyclic adenosine analog (TCN) has shown in vivo antitumor activity and in vitro cyctotoxicity. Other investigators have reported that TCN is phosphorylated by adenosine kinase, but the biochemical mechanism of TCN cytotoxicity has not been elucidated. Preliminary results from the principal investigator's laboratory suggest that incubation of L1210 cells with TCN causes selective killing of cells in S-phase and accumulation of the remaining cells in G1. The proposed research will directly test the cell cycle phase specificity of cell killing by TCN using synchronized cell populations. The possibility that the S-phase toxicity is associated with selective inhibition of DNA synthesis will be investigated by determining the incorporation of (3H)dThd and (3H)Urd at early times of TCN treatment. It is also proposed to investigate whether TCN treatment of cells results in DNA damage. Possible biochemical mechanisms underlying these effects will be studied. Initially, the derivatives of TCN that might be involved in causing the cytotoxic effect will be identified. L1210 cells will be incubated with 14C-TCN, and the metabolites will be separated and identified using high pressure liquid chromatography. The effects of TCN on natural ribonucleotide pools also will be studied in these experiments to determine whether effects at this level play a role in the mechanism of cytotoxicity. Determination of the levels of deoxyribonucleotide triphosphates will provide evidence on the possibility that TCN metabolites may cause inhibition of ribonucleotide reductase. The possible effects of TCN and its metabolites on polyamine levels in L1210 cells will also be investigated.